Synergistic antibacterial activity of medium polarity oils in combination with antibacterial agents on bacterial biofilms

ABSTRACT

The compositions of the present invention comprise at least one medium polarity oil and at least one antibacterial agent, the combination of which produces a synergistic antibacterial effect against bacterial biofilms. Methods are disclosed for the reduction of bacteria in and/or elimination of bacterial biofilms on biological and non-biological surfaces, as well as methods for the treatment of wounds, skin lesions, mucous membrane lesions, and other biological surfaces infected or contaminated with bacterial biofilms.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/529,303 filed Aug. 1, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/754,703 filed Feb. 23, 2018, which is a nationalphase application under 35 U.S.C. § 371 of International Application No.PCT/US2016/048110 filed Aug. 23, 2016, which claims the benefit of U.S.Provisional Application No. 62/209,181 filed Aug. 24, 2015. The contentsof the referenced applications are incorporated into the presentapplication by reference.

FIELD OF THE INVENTION

The present invention generally relates to compositions havingantibacterial activity against bacterial biofilms and use of suchcompositions for the reduction of bacteria in and/or elimination ofbacterial biofilms on biological and non-biological surfaces. Inparticular, the compositions can include a combination of a mediumpolarity oil(s) and an antibacterial agent(s) and can be used to treatwounds, skin lesions, mucous membrane lesions, and other biologicalsurfaces infected or contaminated with bacterial biofilms.

BACKGROUND OF THE INVENTION

Bacterial biofilms are populations of bacteria attached to a surface.Bacteria in a biofilm are frequently embedded within a self-producedmatrix of an extracellular polymeric substance (EPS), which holds thebacteria together in a mass and firmly attaches the bacterial mass tothe underlying surface. The bacterial biofilm EPS, which is oftenreferred to as slime, is a polymeric conglomeration generally composedof extracellular DNA, proteins, polysaccharides, and variousbiopolymers. Biofilms can form on biological or non-biological surfacesand can be prevalent in both industrial and clinical settings.

Evidence has shown that biofilms constitute a significant threat tohuman health. Biofilms are responsible for more than 80% of microbialinfections in the body (“Research on Microbial Biofilms”, NationalInstitutes of Health, PA Number: PA-03-047, Dec. 20, 2002). Biofilms areinvolved in health conditions such as urinary tract infections,cystitis, lung infections, skin infections, mucous membrane infections,sinus infections, ear infections, acne, dental caries, periodontitis,nosocomial infections, open wounds, and chronic wounds. Additionally,biofilms form on medical devices such as: urinary tract prostheses;urinary tract catheters; peritoneal membrane catheters, peritonealdialysis catheters, indwelling catheters for hemodialysis and forchronic administration of chemotherapeutic agents (Hickman catheters);cardiac implants such as pacemakers, prosthetic heart valves,ventricular assist devices, and synthetic vascular grafts and stents;prostheses; percutaneous sutures; and tracheal and ventilator tubing.

Bacteria growing in biofilms exhibit increased tolerance to antibioticsand antibacterial agents and are very difficult to substantially reduceor eliminate. Bacteria within biofilms have increased tolerance (up to1000-fold higher) to antibacterial compounds than bacteria not withinbiofilms, even though these same bacteria are sensitive to these agentsif grown under planktonic conditions (“Research on Microbial Biofilms”,National Institutes of Health, PA Number: PA-03-047, Dec. 20, 2002).Bacteria grown in biofilms are also physiologically distinct from thesame bacteria grown under planktonic conditions. The bacteria inbiofilms are stratified into different metabolic states depending onwhere in the biofilm they reside and thus display different phenotypescompared to their free-living counterparts. Another theory behind theantimicrobial tolerance of bacteria in biofilms is the protective roleof the EPS. The EPS can be visualized as a “mesh” or a network that canphysically prevent foreign agents (i.e., antibacterial agents) fromreaching the bacteria. Because of the EPS, altered metabolic states andacquired resistance factors, biofilms have a multifactorial tolerance toantibacterial agents and antibiotics. Moreover, most of theantibacterial formulations are water-based preparations, making it evenharder for the antibacterial active to penetrate the biofilm network dueto high surface tension of water molecules.

Wounds, mucous membrane lesions, and skin lesions are especiallysusceptible to bacterial infection. From a microbiological perspective,the primary function of normal, intact skin is to control microbialpopulations that live on the skin surface and to prevent underlyingtissue from becoming colonized and invaded by potential pathogens.Exposure of subcutaneous tissue, such as a wound, mucous membranelesion, or skin lesion, provides a moist, warm and nutritiousenvironment that is conducive to microbial colonization andproliferation. Since wound colonization is mostly polymicrobial,involving numerous microorganisms that are potentially pathogenic, anywound, mucous membrane lesion, or skin lesion is at some risk ofbecoming infected.

Wounds often have multiple barriers to healing. Wound healing andinfection is influenced by the relationship between the ability ofbacteria to create a stable, prosperous community within a woundenvironment and the ability of the host to control the bacterialcommunity. Since bacteria are rapidly able to form their own protectivemicroenvironment, i.e., a biofilm, following their attachment to asurface, the ability of the host to control these organisms is likely todecrease as the biofilm community matures, ultimately affecting theability of the wound to heal. Wounds in which healing is delayed, i.e.,chronic wounds, are of particular concern with respect to biofilmformation. Some have linked biofilms to chronic wounds (Mertz, 2003,Wounds, 15: 1-9). Wounds such as diabetic foot ulcers, venous ulcers,arterial ulcers, decubitus ulcers, stasis ulcers, pressure ulcers, andburns are examples of wounds which may become chronic wounds. Bacterialbiofilms in chronic wounds are generally not resolved by the host'simmune system, and these biofilms have an increased tolerance tosystemic and topical antibacterial/antibiotic agents. Accordingly,bacterial biofilm infections in chronic wounds are very difficult tosubstantially reduce or eliminate.

Particularly virulent organisms in wounds, mucous membrane lesions, andskin lesions are gram-positive bacteria such as staphylococcus spp.,streptococcus spp., and enterococci spp. Biofilms of Staphylococcusaureus, including resistant strains such as methicillin resistantstaphylococcus aureus (MRSA), have become increasingly problematic inwounds, skin lesions, and mucous membrane lesions. These organisms,especially MRSA, can reside in the anterior nares and cause lesions inthe nose which can also spread to other parts of the body, causing skinlesions and mucous membrane lesions at those sites. The gram-negativebacteria Pseudomonas aeruginosa is also a particularly virulent organismin wounds (Bjarnsholt, 2008, Wound Repair and Regeneration; andJacobsen, 2011, International Wound Journal).

In recent years, there have been numerous efforts to use variousantibiotics and antibacterial agents for the treatment of mucousmembrane lesions, skin lesions and chronic wounds, many of which areinfected or contaminated with bacterial biofilms. These agents are ofvarying chemical compounds and include, among others, peptides such asvancomycin, and antibacterial agents such as mupirocin, iodinecompounds, and silver/silver ions. However, many bacteria have becomeincreasingly resistant to these compounds.

Thus, there is a need for safe and effective compositions which canreduce bacteria in or eliminate bacterial biofilms in wounds, mucousmembrane lesions, and skin lesions, and on other biological andnon-biological surfaces.

SUMMARY OF THE INVENTION

The present invention provides a solution to the aforementionedlimitations and deficiencies in the art relating to bacterial biofilms.In particular, the solution is premised on the combination of at leastone medium polarity oil having an octanol-water partition coefficient(log P) of 0.5 to 2.0 and at least one antibacterial agent in acomposition. Surprisingly, this combination produces a synergisticantibacterial effect against bacterial biofilms. Stated another way, thesynergistic effect means the total antibacterial activity againstbacterial biofilms of the combination of the two components, i.e., themedium polarity oil plus the antibacterial agent, is greater than thesum of the antibacterial activity against biofilms of each componentwhen measured separately. Without being bound by theory, it ispostulated that the medium polarity oils, being dispersible in both oiland aqueous media, enhance penetration through the extracellularpolymeric substance (EPS) produced by the biofilm bacteria, and increasethe targeted delivery of the antibacterial agent. This combination of atleast one medium polarity oil and at least one antibacterial agent canbe used to produce a composition capable of: treating wounds, mucousmembrane lesions, skin lesions, and/or other biological surfacesinfected or contaminated with bacterial biofilms; reducing bacteria inand/or eliminating bacterial biofilms on biological surfaces; and/orreducing bacteria in and/or eliminating bacterial biofilms onnon-biological surfaces such as on medical devices.

In one aspect of the invention, disclosed are methods of treating awound, mucous membrane lesion, or skin lesion infected or contaminatedwith a bacterial biofilm, the method comprising topically administeringto the wound, mucous membrane lesion, or skin lesion a compositioncomprising a combination of at least one medium polarity oil having anoctanol-water partition coefficient (log P) of 0.5 to 2.0 and at leastone antibacterial agent, wherein the combination of the at least onemedium polarity oil and the at least one antibacterial agent exhibitssynergistic antibacterial activity against the biofilm.

In another aspect of the invention, disclosed are methods of reducingbacteria in or eliminating a bacterial biofilm on a biological surface,the method comprising administering to the biological surface acomposition comprising a combination of at least one medium polarity oilhaving an octanol-water partition coefficient (log P) of 0.5 to 2.0 andat least one antibacterial agent, wherein the combination of the atleast one medium polarity oil and the at least one antibacterial agentexhibits synergistic antibacterial activity against the biofilm.

In still another aspect of the invention, disclosed are methods ofreducing bacteria in or eliminating a bacterial biofilm on anon-biological surface, the method comprising administering to thenon-biological surface a composition comprising a combination of atleast one medium polarity oil having an octanol-water partitioncoefficient (log P) of 0.5 to 2.0 and at least one antibacterial agent,wherein the combination of the at least one medium polarity oil and theat least one antibacterial agent exhibits synergistic antibacterialactivity against the biofilm.

In another aspect of the invention, disclosed are compositions suitablefor application to biological and non-biological surfaces havingbacterial biofilms, comprising a combination of at least one mediumpolarity oil having an octanol-water partition coefficient (log P) of0.5 to 2.0 and at least one antibacterial agent, wherein theconcentrations of the at least one medium polarity oil and the at leastone antibacterial agent in the composition are at amounts that exhibitsynergistic antibacterial activity against bacterial biofilms onbiological and non-biological surfaces.

In still another aspect of the invention, disclosed are articles ofmanufacture comprising a surface coated with the compositions of theinvention.

Also disclosed in the context of the present invention are embodiments 1to 126.

Embodiment 1: A method of treating a wound, mucous membrane lesion, orskin lesion infected or contaminated with a bacterial biofilm, themethod comprising topically administering to the wound, mucous membranelesion, or skin lesion a composition comprising a combination of atleast one medium polarity oil having an octanol-water partitioncoefficient (log P) of 0.5 to 2.0 and at least one antibacterial agent,wherein the combination of the at least one medium polarity oil and theat least one antibacterial agent exhibits synergistic antibacterialactivity against the biofilm.

Embodiment 2: The method of embodiment 1, wherein the compositionfurther comprises a carrier suitable for topical treatment.

Embodiment 3: The method of embodiments 1 or 2, wherein theantibacterial agent is a silver compound, an iodine compound, or anantibiotic.

Embodiment 4: The method of embodiments 1 or 2, wherein theantibacterial agent is not a C9-C12 aliphatic alcohol. In some aspects,the composition in embodiment 4 is free of/does not include a C9-C12aliphatic alcohol.

Embodiment 5: The method of embodiments 1 or 2, wherein theantibacterial agent is a silver compound.

Embodiment 6: The method of embodiment 5, wherein the compositioncomprises a carrier suitable for topical treatment and the carrier is aringing gel.

Embodiment 7: The method of embodiments 5 or 6, wherein the silvercompound is silver sulfadiazine, silver nitrate, or silver chloride.

Embodiment 8: The method of embodiments 1 or 2, wherein theantibacterial agent is an iodine compound.

Embodiment 9: The method of embodiment 8, wherein the iodine compound isan iodophor.

Embodiment 10: The method of embodiment 9, wherein the iodophor iscadexomer-iodine.

Embodiment 11: The method of embodiment 9, wherein the iodophor ispovidone-iodine.

Embodiment 12: The method of embodiments 1 or 2, wherein theantibacterial agent is an antibiotic.

Embodiment 13: The method of embodiment 12, wherein the compositioncomprises a carrier suitable for topical treatment and the carrier is aringing gel.

Embodiment 14: The method of embodiments 12 or 13, wherein theantibiotic is an aminoglycoside antibiotic.

Embodiment 15: The method of embodiment 14, wherein the aminoglycosideantibiotic is gentamicin or gentamicin sulfate.

Embodiment 16: The method of embodiments 12 or 13, wherein theantibiotic is a polypeptide antibiotic.

Embodiment 17: The method of embodiment 16, wherein the polypeptideantibiotic is colistin or colistin sulfate.

Embodiment 18: The method of any one of embodiments 1 to 17, wherein themedium polarity oil is an ester.

Embodiment 19: The method of any one of embodiments 1 to 18, wherein themedium polarity oil is a fatty acid ester.

Embodiment 20: The method of any one of embodiments 1 to 19, wherein themedium polarity oil is a glyceryl ester.

Embodiment 21: The method of embodiment 20, wherein the glyceryl esteris glyceryl caprylate/caprate.

Embodiment 22: The method of embodiment 21, wherein the glycerylcaprylate/caprate is at a concentration of 7 to 40% w/w.

Embodiment 23: The method of embodiment 21, wherein the glycerylcaprylate/caprate is at a concentration of 7 to 15% w/w.

Embodiment 24: The method of any one of embodiments 1 to 23, wherein thebacterial biofilm is a gram-positive bacterial biofilm.

Embodiment 25: The method of embodiment 24, wherein the gram-positivebacterial biofilm is a Staphylococcus sp.

Embodiment 26: The method of embodiment 25, wherein the Staphylococcussp. is Staphylococcus aureus.

Embodiment 27: The method of embodiment 26, wherein the Staphylococcussp. is methicillin resistant Staphylococcus aureus (MRSA).

Embodiment 28: The method of any one of embodiments 1 to 23, wherein thebacterial biofilm is a gram-negative bacterial biofilm.

Embodiment 29: The method of embodiment 28, wherein the gram-negativebacterial biofilm is a Pseudomonas sp.

Embodiment 30: The method of embodiment 29, wherein the Pseudomonas sp.is Pseudomonas aeruginosa.

Embodiment 31: The method of any one of embodiments 1 to 30, wherein thewound is a chronic wound.

Embodiment 32: The method of embodiment 31, wherein the chronic wound isa diabetic foot ulcer, venous ulcer, arterial ulcer, decubitus ulcer,stasis ulcer, pressure ulcer, or burn.

Embodiment 33: The method of any one of embodiments 1 to 30, wherein theskin lesion or mucous membrane lesion, is a blister, ulceration,abrasion, wart, scrape, or infection.

Embodiment 34: The method of any one of embodiments 1 to 33, wherein theconcentrations of the at least one medium polarity oil and the at leastone antibacterial agent in the composition are at amounts that exhibitsynergistic antibacterial activity against the biofilm.

Embodiment 35: The method of any one of embodiments 1 to 34, wherein theoctanol-water partition coefficient (log P) is experimentally determinedby the ASTM Standard Test Method for Partition Coefficient(N-Octanol/Water) Estimation by Liquid Chromatography, Designation E1147-92.

Embodiment 36: A method of reducing bacteria in or eliminating abacterial biofilm on a biological surface, the method comprisingadministering to the biological surface a composition comprising acombination of at least one medium polarity oil having an octanol-waterpartition coefficient (log P) of 0.5 to 2.0 and at least oneantibacterial agent, wherein the combination of the at least one mediumpolarity oil and the at least one antibacterial agent exhibitssynergistic antibacterial activity against the biofilm.

Embodiment 37: The method of embodiment 36, wherein the compositionfurther comprises a carrier.

Embodiment 38: The method of embodiments 36 or 37, wherein theantibacterial compound is a silver compound, an iodine compound, or anantibiotic.

Embodiment 39: The method of embodiments 36 or 37, wherein theantibacterial agent is not a C9-C12 aliphatic alcohol. In some aspects,the composition in embodiment 39 is free of/does not include a C9-C12aliphatic alcohol.

Embodiment 40: The method of embodiments 36 or 37, wherein theantibacterial agent is a silver compound.

Embodiment 41: The method of embodiment 40, wherein the compositioncomprises a carrier and the carrier is a ringing gel.

Embodiment 42: The method of embodiments 40 or 41, wherein the silvercompound is silver sulfadiazine, silver nitrate, or silver chloride.

Embodiment 43: The method of embodiments 36 or 37, wherein theantibacterial agent is an iodine compound.

Embodiment 44: The method of embodiment 43, wherein the iodine compoundis an iodophor.

Embodiment 45: The method of embodiment 44, wherein the iodophor iscadexomer-iodine.

Embodiment 46: The method of embodiment 44, wherein the iodophor ispovidone-iodine.

Embodiment 47: The method of embodiments 36 or 37, wherein theantibacterial agent is an antibiotic.

Embodiment 48: The method of embodiment 47, wherein the compositioncomprises a carrier and the carrier is a ringing gel.

Embodiment 49: The method of embodiments 47 or 48, wherein theantibiotic is an aminoglycoside antibiotic.

Embodiment 50: The method of embodiment 49, wherein the aminoglycosideantibiotic is gentamicin or gentamicin sulfate.

Embodiment 51: The method of embodiments 47 or 48, wherein theantibiotic is a polypeptide antibiotic.

Embodiment 52: The method of embodiment 51, wherein the polypeptideantibiotic is colistin or colistin sulfate.

Embodiment 53: The method of any one of embodiments 36 to 52, whereinthe medium polarity oil is an ester.

Embodiment 54: The method of any one of embodiments 36 to 53, whereinthe medium polarity oil is a fatty acid ester.

Embodiment 55: The method of any one of embodiments 36 to 54, whereinthe medium polarity oil is a glyceryl ester.

Embodiment 56: The method of embodiment 55, wherein the glyceryl esteris glyceryl caprylate/caprate.

Embodiment 57: The method of embodiment 56, wherein the glycerylcaprylate/caprate is at a concentration of 7 to 40% w/w.

Embodiment 58: The method of embodiment 56, wherein the glycerylcaprylate/caprate is at a concentration of 7 to 15% w/w.

Embodiment 59: The method of any one of embodiments 36 to 58, whereinthe bacterial biofilm is a gram-positive bacterial biofilm.

Embodiment 60: The method of embodiment 59, wherein the gram-positivebacterial biofilm is a Staphylococcus sp.

Embodiment 61: The method of embodiment 60, wherein the Staphylococcussp. is Staphylococcus aureus.

Embodiment 62: The method of embodiment 61, wherein the Staphylococcussp. is methicillin resistant Staphylococcus aureus (MRSA).

Embodiment 63: The method of any one of embodiments 36 to 58, whereinthe bacterial biofilm is a gram-negative bacterial biofilm.

Embodiment 64: The method of embodiment 63, wherein the gram-negativebacterial biofilm is a Pseudomonas sp.

Embodiment 65: The method of embodiment 64, wherein the Pseudomonas sp.is Pseudomonas aeruginosa.

Embodiment 66: The method of any one of embodiments 36 to 65, whereinthe biological surface is a chronic wound.

Embodiment 67: The method of embodiment 66, wherein the chronic wound isa diabetic foot ulcer, venous ulcer, arterial ulcer, decubitus ulcer,stasis ulcer, pressure ulcer, or burn.

Embodiment 68: The method of any one of embodiments 36 to 65, whereinthe biological surface is a skin lesion, or mucous membrane lesion.

Embodiment 69: The method of embodiment 68, wherein the skin lesion ormucous membrane lesion, is a blister, ulceration, abrasion, wart,scrape, or infection.

Embodiment 70: The method of any one of embodiments 36 to 69, whereinthe concentrations of the at least one medium polarity oil and the atleast one antibacterial agent in the composition are at amounts thatexhibit synergistic antibacterial activity against the biofilm.

Embodiment 71: The method of any one of embodiments 36 to 70, whereinthe octanol-water partition coefficient (log P) is experimentallydetermined by the ASTM Standard Test Method for Partition Coefficient(N-Octanol/Water) Estimation by Liquid Chromatography, Designation E1147-92.

Embodiment 72: A method of reducing bacteria in or eliminating abacterial biofilm on a non-biological surface, the method comprisingadministering to the non-biological surface a composition comprising acombination of at least one medium polarity oil having an octanol-waterpartition coefficient (log P) of 0.5 to 2.0 and at least oneantibacterial agent, wherein the combination of the at least one mediumpolarity oil and the at least one antibacterial agent exhibitssynergistic antibacterial activity against the biofilm.

Embodiment 73: The method of embodiment 72, wherein the compositionfurther comprises a carrier suitable for application to a non-biologicalsurface.

Embodiment 74: The method of embodiments 72 or 73, wherein thenon-biological surface is a medical device.

Embodiment 75: The method of embodiment 74, wherein the medical deviceis a urinary tract prosthesis, urinary tract catheter, peritonealmembrane catheter, peritoneal dialysis catheter, indwelling catheter forhemodialysis, indwelling catheter for administration of chemotherapeuticagents, cardiac implant, pacemaker, prosthetic heart valve, ventricularassist device, synthetic vascular graft, synthetic vascular stent,prosthesis, percutaneous suture, tracheal tubing, or ventilator tubing.

Embodiment 76: The method of any one of embodiments 72 to 75, whereinthe concentrations of the at least one medium polarity oil and the atleast one antibacterial agent in the composition are at amounts thatexhibit synergistic antibacterial activity against the biofilm.

Embodiment 77: The method of any one of embodiments 72 to 76, whereinthe octanol-water partition coefficient (log P) is experimentallydetermined by the ASTM Standard Test Method for Partition Coefficient(N-Octanol/Water) Estimation by Liquid Chromatography, Designation E1147-92.

Embodiment 78: A composition comprising a combination of at least onemedium polarity oil having an octanol-water partition coefficient (logP) of 0.5 to 2.0 and at least one antibacterial agent, wherein theconcentrations of the at least one medium polarity oil and the at leastone antibacterial agent in the composition are at amounts that exhibitsynergistic antibacterial activity against bacterial biofilms onbiological and non-biological surfaces.

Embodiment 79: The composition of embodiment 78, wherein the compositionfurther comprises a carrier.

Embodiment 80: The composition of embodiment 79, wherein the carrier isa carrier suitable for application to a non-biological surface.

Embodiment 81: The composition of embodiment 79, wherein the carrier isa pharmaceutical carrier.

Embodiment 82: The composition of embodiment 81, wherein thepharmaceutical carrier is a lotion, solution, suspension, liquid,emulsion, cream, gel, ringing gel, ointment, paste, aerosol spray,aerosol foam, non-aerosol spray, non-aerosol foam, film, or sheet.

Embodiment 83: The composition of any one of embodiments 78 to 82,wherein the antibacterial agent is a silver compound, an iodinecompound, or an antibiotic.

Embodiment 84: The composition of any one of embodiments 78 to 82,wherein the antibacterial agent is not a C9-C12 aliphatic alcohol. Insome aspects, the composition in embodiment 84 is free of/does notinclude a C9-C12 aliphatic alcohol.

Embodiment 85: The composition of any one of embodiments 78 to 82,wherein the antibacterial agent is a silver compound.

Embodiment 86: The composition of embodiment 85, wherein the compositioncomprises a carrier and the carrier is a ringing gel.

Embodiment 87: The composition of embodiments 85 or 86, wherein thesilver compound is silver sulfadiazine, silver nitrate, or silverchloride.

Embodiment 88: The composition of any of embodiments 85 to 87, whereinthe silver compound is at a concentration of 0.1 to 5% w/w.

Embodiment 89: The composition of any of embodiments 78 to 82, whereinthe antibacterial agent is an iodine compound.

Embodiment 90: The composition of embodiment 89, wherein the iodinecompound is an iodophor.

Embodiment 91: The composition of embodiment 90, wherein the iodophor iscadexomer-iodine.

Embodiment 92: The composition of embodiment 91, wherein thecadexomer-iodine is at a concentration of 40 to 60% w/w.

Embodiment 93: The composition of embodiment 90, wherein the iodophor ispovidone-iodine.

Embodiment 94: The composition of embodiment 93, wherein thepovidone-iodine is at a concentration of 1 to 20% w/w.

Embodiment 95: The composition of any one of embodiments 78 to 82,wherein the antibacterial agent is an antibiotic.

Embodiment 96: The composition of embodiment 95, wherein the compositioncomprises a carrier and the carrier is a ringing gel.

Embodiment 97: The composition of embodiments 95 or 96, wherein theantibiotic is an aminoglycoside antibiotic.

Embodiment 98: The composition of embodiment 97, wherein theaminoglycoside antibiotic is gentamicin or gentamicin sulfate.

Embodiment 99: The composition of embodiment 98, wherein the gentamicinor gentamicin sulfate is at a concentration of 0.1 to 5% w/w.

Embodiment 100: The composition of embodiments 95 or 96, wherein theantibiotic is a polypeptide antibiotic.

Embodiment 101: The composition of embodiment 100, wherein thepolypeptide antibiotic is colistin or colistin sulfate.

Embodiment 102: The composition of embodiment 101, wherein theconcentration of colistin or colistin sulfate is 0.01 to 2% w/w.

Embodiment 103: The composition of any one of embodiments 78 to 102,wherein the medium polarity oil is an ester.

Embodiment 104: The composition of any one of embodiments 78 to 103,wherein the medium polarity oil is a fatty acid ester.

Embodiment 105: The composition of any one of embodiments 78 to 104,wherein the medium polarity oil is a glyceryl ester.

Embodiment 106: The composition of embodiment 105, wherein the glycerylester is glyceryl caprylate/caprate.

Embodiment 107: The composition of embodiment 106, wherein the glycerylcaprylate/caprate is at a concentration of 7 to 40% w/w.

Embodiment 108: The composition of embodiment 106, wherein the glycerylcaprylate/caprate is at a concentration of 7 to 15% w/w.

Embodiment 109: The composition of any one of embodiments 78 to 108,wherein the bacterial biofilm is a gram-positive bacterial biofilm.

Embodiment 110: The composition of embodiment 109, wherein thegram-positive bacterial biofilm is a Staphylococcus sp.

Embodiment 111: The composition of embodiment 110, wherein theStaphylococcus sp. is Staphylococcus aureus.

Embodiment 112: The composition of embodiment 111, wherein theStaphylococcus sp. is methicillin resistant Staphylococcus aureus(MRSA).

Embodiment 113: The composition of any one of embodiments 78 to 108,wherein the bacterial biofilm is a gram-negative bacterial biofilm.

Embodiment 114: The composition of embodiment 113, wherein thegram-negative bacterial biofilm is a Pseudomonas sp.

Embodiment 115: The composition of embodiment 114, wherein thePseudomonas sp. is Pseudomonas aeruginosa.

Embodiment 116: The composition of any one of embodiments 78 to 115,wherein the biological surface is a chronic wound.

Embodiment 117: The composition of embodiment 116, wherein the chronicwound is a diabetic foot ulcer, venous ulcer, arterial ulcer, decubitusulcer, stasis ulcer, pressure ulcer, or burn.

Embodiment 118: The composition of any one of embodiments 78 to 115,wherein the biological surface is a skin lesion, or mucous membranelesion.

Embodiment 119: The composition of embodiment 118, wherein the skinlesion or mucous membrane lesion, is a blister, ulceration, abrasion,wart, scrape, or infection.

Embodiment 120: The composition of any one of embodiments 78 to 119,wherein the octanol-water partition coefficient (log P) isexperimentally determined by the ASTM Standard Test Method for PartitionCoefficient (N-Octanol/Water) Estimation by Liquid Chromatography,Designation E 1147-92.

Embodiment 121: An article of manufacture comprising a surface coatedwith the composition of any one of embodiments 78 to 115.

Embodiment 122: The article of manufacture of embodiment 121, whereinthe article of manufacture is a medical device.

Embodiment 123: The article of manufacture of embodiment 121, whereinthe medical device is a urinary tract prosthesis, urinary tractcatheter, peritoneal membrane catheter, peritoneal dialysis catheter,indwelling catheter for hemodialysis, indwelling catheter foradministration of chemotherapeutic agents, cardiac implant, pacemaker,prosthetic heart valve, ventricular assist device, synthetic vasculargraft, synthetic vascular stent, prosthesis, percutaneous suture,tracheal tubing, or ventilator tubing.

Embodiment 124: The article of manufacture of any one of embodiments 121to 123, wherein a biofilm is not present on the coated surface.

Embodiment 125: The article of manufacture of any one of embodiments 121to 123, wherein a biofilm is present on the coated surface.

Embodiment 126: The article of manufacture of any one of embodiments 121to 125, wherein the octanol-water partition coefficient (log P) isexperimentally determined by the ASTM Standard Test Method for PartitionCoefficient (N-Octanol/Water) Estimation by Liquid Chromatography,Designation E 1147-92.

Unless otherwise specified, the percent values expressed herein areweight by weight and are in relation to the weight of the totalcomposition. By way of example, 10 grams of an ingredient in 100 gramsof a composition that includes the 10 grams of the ingredient is 10 wt.% of the ingredient in the composition.

The term “reduce,” “reduced,” “reducing,” or “reduction” in the contextof a bacterial biofilm means a reduction in the count of bacteriapresent in the biofilm.

The term “treat,” “treated,” or “treating,” in the context of treating abacterial biofilm on a biological surface, or treating a mucous membranelesion, a wound, or a skin lesion, means any measurable decrease orcomplete elimination of the bacterial biofilm, and/or a therapeuticimprovement of the mucous membrane lesion, wound, or skin lesion.

The term “effective,” in the context of treating a bacterial biofilm ortreating a wound, mucous membrane lesion, or skin lesion means adequateto accomplish a desired, expected, or intended result, including atherapeutic improvement.

The term “eliminate,” “eliminated,” “eliminating,” or “elimination” inthe context of a bacterial biofilm means total eradication of thebacteria present in the biofilm.

The term “wound” as used herein means an external wound of the skin ormucous membranes and includes chronic and acute wounds.

The terms “about” or “approximately” are defined as being close to asunderstood by one of ordinary skill in the art, and in one non-limitingembodiment the terms are defined to be within 10%, preferably within 5%,more preferably within 1%, and most preferably within 0.5%.

The words “comprising” (and any form of comprising, such as “comprise”and “comprises”), “having” (and any form of having, such as “have” and“has”), “including” (and any form of including, such as “includes” and“include”) or “containing” (and any form of containing, such as“contains” and “contain”) are inclusive or open-ended and do not excludeadditional, unrecited elements or method steps.

The use of the word “a” or “an” when used in conjunction with the terms“comprising,” “having,” “including,” or “containing” (or any variationsof these words) may mean “one,” but it is also consistent with themeaning of “one or more,” “at least one,” and “one or more than one.”

The compositions and methods for their use can “comprise,” “consistessentially of,” or “consist of” any of the ingredients or stepsdisclosed throughout the specification.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions of the inventioncan be used to achieve methods of the invention.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the log reduction of bacteria in an in-vitro S. aureusbiofilm model after treatment with various oils of various polarity vs.moist control.

FIG. 2 shows the comparison of the retention times of the referencestandards to the retention time of CAPMUL MCM to calculate the log Pvalue of CAPMUL MCM as per ASTM method.

FIG. 3 shows the log reduction of bacteria in the P. aeruginosa biofilmmodel after treatment with the treatment formulations from Example 2(cadexomer iodine based formulations) vs. moist control.

FIG. 4 shows the log reduction of bacteria in the P. aeruginosa biofilmmodel after treatment with the treatment formulations from Example 2(silver chloride based formulations) vs. moist control.

FIG. 5 shows the log reduction of bacteria in the P. aeruginosa biofilmmodel after treatment with the treatment formulations from Example 2(other silver based formulations) vs. moist control.

FIG. 6 shows the log reduction of bacteria in the P. aeruginosa biofilmmodel after treatment with the treatment formulations from Example 2(povidone-iodine based formulations) vs. moist control.

FIG. 7 shows the log reduction of bacteria in the P. aeruginosa biofilmmodel after treatment with the treatment formulations from Example 2(gentamicin based formulations) vs. moist control.

FIG. 8 shows the log reduction of bacteria in the P. aeruginosa biofilmmodel after treatment with the treatment formulations from Example 2(colistin based formulations) vs. moist control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions useful for thereduction of bacteria in and/or elimination of bacterial biofilms onsurfaces. In particular, the present invention provides compositionswhich exhibit activity against bacterial biofilms, and methods of usingthe same to treat biological and non-biological surfaces infected orcontaminated with bacterial biofilms by reducing bacteria in oreliminating the bacterial biofilm. In one aspect, the present inventionrelates to methods and compositions useful for the treatment of wounds,skin lesions, mucous membrane lesions, and other biological surfacesinfected or contaminated with bacterial biofilms. In another aspect, thepresent invention relates to methods and compositions useful for thereduction of bacteria in and/or elimination of bacterial biofilms onnon-biological surfaces such as medical devices.

The compositions of the present invention comprise at least one mediumpolarity oil and at least one antibacterial agent. Surprisingly, thecombination of at least one medium polarity oil and at least oneantibacterial agent produces a synergistic antibacterial effect againstbacterial biofilms. Stated another way, the total antibacterial activityagainst bacterial biofilms of the combination of the two components,i.e., the medium polarity oil plus the antibacterial agent, is greaterthan the sum of the antibacterial activity against biofilms of eachcomponent when measured separately.

I. COMPOSITIONS

The compositions of the present invention comprise at least one mediumpolarity oil and at least one antibacterial agent, both of which aredescribed below in a non-limiting manner. The concentrations of the atleast one medium polarity oil and the at least one antibacterial agentin the compositions are at an amount that exhibits synergisticantibacterial activity against bacterial biofilms.

The compositions of the invention can also include an acceptable carriersuch as a carrier suitable for topical products or a carrier suitablefor application to a non-biological surface, such as a medical device.The carrier may also be a pharmaceutical carrier suitable forapplication to biological surfaces including topical surfaces. Thecompositions of the present invention can comprise carriers suitable fortopical treatment of skin, mucous membranes, and wounds. Non-limitingexamples of carriers include lotions, solutions, suspensions, liquids,emulsions, creams, gels, ringing gels, ointments, pastes, aerosolsprays, aerosol foams, non-aerosol sprays, non-aerosol foams, films,powders, and sheets. The compositions can be impregnated in gauzes,bandages, or other wound dressing materials. Non-limiting examples ofcarriers suitable for topical treatment of skin, mucous membranes andwounds include those disclosed in U.S. Pat. No. 6,399,092, hereinincorporated by reference. Other examples of carriers suitable fortopical treatment of skin, mucous membranes, and wounds includepolyethylene glycol ointments. Especially suitable carriers includeringing gels, which are viscous microemulsions that are generallytransparent, and exhibit a ringing phenomenon when excited to mechanicalvibrations. Ringing gels can be O/W or W/O. Ringing gels are inherentlyviscous and do not need the addition of thickening agents in order toprovide a viscous composition. Formulations of ringing gels are known inthe art. An example of a formulation of a ringing gel carrier compriseswater, a glyceryl ester such as CAPMUL® MCM, and a poloxamer such aspoloxamer-407. Ringing gels are especially suitable for topicalapplications, such as to wounds or skin, in that the viscous compositionwill not run out of a wound or run off the skin when applied. Anadditional benefit of the use of ringing gel carriers for the presentinvention is that since thickening agents are not needed, there is lesschance of ingredients interfering with the synergistic activity of thecomposition and less chance for irritation when applied to biologicalsurfaces such as skin or wounds. Viscosity values for ringing gelcarriers of the present invention can be at least 6250 cps, or at least12,500 cps, or at least 25,000 cps, or at least 50,000 cps, or at least60,000 cps, or at least 75,000 cps, or 6250 cps to 125,000 cps, or12,500 cps to 125,000 cps, or 25,000 cps to 125,000 cps, or 50,000 cpsto 125,000 cps, or 60,000 cps to 125,000 cps, or 75,000 cps to 125,000cps when measured with a Brookfield viscometer with a small sampleadapter, spindle #14, 6R chamber, at room temperature (22°-25 ° C.), at10 rpm for 1 minute.

The compositions of the invention may further comprise functionalingredients suitable for use in compositions for application tobiological surfaces or non-biological surfaces. Non-limiting examplesinclude absorbents, super absorbents, antibacterial agents,antioxidants, binders, buffering agents, bulking agents, chelatingagents, colorants, biocides, deodorant agents, emulsion stabilizers,film formers, fragrance ingredients, humectants, lytic agents, enzymaticagents, opacifying agents, oxidizing agents, pH adjusters, plasticizers,preservatives, reducing agents, emollient skin conditioning agents,humectant skin conditioning agents, moisturizers, surfactants,emulsifying agents, cleansing agents, foaming agents, hydrotopes,solvents, suspending agents, viscosity control agents (rheologymodifiers), viscosity increasing agents (thickeners), and propellants.Listings and monographs of suitable functional ingredients are disclosedin McCutcheon's Vol. 1 Emulsifiers & Detergents, and Vol. 2 FunctionalMaterials, 2001, herein incorporated by reference.

The compositions of the invention can further comprise pharmaceuticallyactive ingredients, cosmetically active ingredients, and vulneraryagents suitable for topical use. The compositions can be sterile orpreserved with preservatives. In some embodiments, the compositions donot include C9-C12 aliphatic alcohols. In some embodiments, thecompositions do not include organic acids. In some embodiments, thecompositions do not include glyceryl monolaurate.

The compositions of the present invention may be packaged in anysuitable package configuration. Non-limiting examples include bottles,lotion pumps, toddles, tubes, jars, non-aerosol pump sprayers, aerosolcontainers, pouches, and packets. The packages may be configured forsingle-use or multiple-use administration.

A. Medium Polarity Oils

The compositions of the invention comprise at least one medium polarityoil with an octanol-water partition coefficient (log P) of 0.5 to 2.0.In some embodiments the octanol-water partition coefficient (log P) is0.7 to 1.9. In other embodiments, the octanol-water partitioncoefficient (log P) is 0.7 to 1.8. The octanol-water partitioncoefficient, “K_(ow)”, also represented as “P”, is the ratio of theequilibrium molar concentration of a chemical in n-octanol and water, indilute solution at a given temperature. The value is usually expressedas the decadic logarithm of this coefficient represented as, “logK_(ow)”, also represented as “log P”. The octanol-water partitioncoefficient is a measure of the hydrophobicity and hydrophilicity of asubstance. Non-polar (hydrophobic) compounds have a high log P whereaspolar (hydrophilic) compounds have a low log P. Medium polaritycompounds have a log P in between non-polar and polar compounds. For thepurposes of the present invention, medium polarity compounds have a logP of 0.5 to 2.0, or 0.7 to 1.9, or 0.7 to 1.8. For the purposes of thepresent invention, the term “medium polarity oil” is usedinterchangeably with the term “medium polarity compound,” and means acompound that is liquid or solid at room temperature having a log P of0.5 to 2.0, or 0.7 to 1.9, or 0.7 to 1.8.

Many methods exist for determining the octanol-water partitioncoefficient of a substance. However, for purposes of the presentinvention, the experimental determination of an octanol-water partitioncoefficient value utilizes a reverse phase (RP) HPLC method. One suchmethod is described in the ASTM Standard Test Method for PartitionCoefficient (N-Octanol/Water) Estimation by Liquid Chromatography,Designation E 1147-92 (Reapproved 2005), herein incorporated byreference. The methodology of the ASTM method is as follows. The testsubstance (solute) is injected onto a liquid chromatograph columncontaining a solid-phase support onto which a commercially availablelong-chain hydrocarbon (for example C8 or C18) has been bonded.Chemicals injected onto such a column move along it by partitioningbetween the mobile phase and the stationary hydrocarbon phase. Amethanol/water solvent system is typically used to elute the solutewhich is subsequently analyzed using an ultraviolet/visible absorptiondetector, refractive index detector, electro-chemical detector, or otherappropriate detector. If the test substance is not amenable to detectionby the available LC detectors, the analyst may collect fractions of thecolumn effluent and analyze for the test substance using gaschromatography, liquid scintillation, or other appropriate technique.The K_(ow) of the test compound is estimated from a linear regressionequation developed from a plot of log (t_(R)−t_(o)) versus log K_(ow),using data determined in a calibration step that involves injecting intothe chromatograph a mixture of reference chemicals. A calibration graphof log (t_(R)−t_(o)) versus log K_(ow) is developed for a number ofreference compounds (typically between 5 and 10) which are structurallysimilar to the test chemical. Lists of values of measured log K_(ow) areavailable for many chemicals. If data on the partition coefficients ofstructurally related compounds are not available, a more generalcalibration graph can be developed using other reference compounds. Thereference compound or test chemical retention time (t_(R)) is the timefrom sample injection to maximum concentration (peak height) of elutedreference compound or test chemical. The internal standard retentiontime (t_(o)) is the time from sample injection to the maximumconcentration (peak height) of the eluted internal standard. Thenormalized retention time for each unknown is t_(R)−t_(o). The resultsare calculated and reported as follows. Using the plot of log(t_(R)−t_(o)) versus log K_(ow) for the reference compounds, compute thelinear regression equation of the form log K_(ow)=a log (t_(R)−t_(o))+b,where a and b are the slope and intercept, respectively. From thestandard curve or regression equation, calculate an estimated log K_(ow)for the test compound corresponding to the measured log (t_(R)−t_(o)).Report the standard curve of log (t_(R)−t_(o)) versus log K_(ow) foreach buffered or unbuffered eluent, or report the regression equation inthe form of log K_(ow)=a log (t_(R)−t_(o))+b. In some embodiments, theoctanol-water partition coefficient (log P) is experimentally determinedby the ASTM Standard Test Method for Partition Coefficient(N-Octanol/Water) Estimation by Liquid Chromatography, Designation E1147-92.

Alternatively, for purposes of the present invention, the octanol-waterpartition coefficient values (log P) may be obtained from the“preferred” or “good” values defined and listed in Exploring QSAR, Vol.1 Fundamentals and Applications in Chemistry and Biology, and Vol. 2,Hydrophobic, Electronic, and Steric Constants, Corwin Hansch, ACSProfessional Reference Book, 1995, herein incorporated by reference.

The inventors determined experimentally that medium polarity oils. i.e.,oils with a log P of 0.5 to 2.0, exhibited some antibacterial activityagainst biofilms, whereas non-polar and polar oils generally did notexhibit very much antibacterial activity against biofilms. The logreduction of bacteria in an in-vitro S. aureus biofilm model aftertreatment with various oils of various polarity vs. moist control isshown in FIG. 1. As can be seen in FIG. 1, the compounds having a log Poutside the range of 0.5 to 2.0 did not exhibit very much antimicrobialactivity against biofilms compared to the compounds having a log Pwithin the range of 0.5 to 2.0. The compounds having a log P outside therange of 0.5 to 2.0 include glyceryl triacetate, methyl benzoate,diethyl acetate, MONOMULS® (glyceryl monolaurate), isopropyl myristate,and oleic acid. Compounds having a log P within the range of 0.5 to 2.0include resorcinol, anisyl alcohol, benzoic acid, benzyl alcohol, ethylacetate, ethyl gallate, phenoxyethanol, phenyethanol, propyl gallate,and glyceryl caprylate/caprate, and are shown in Table 1 below.

In some embodiments, the medium polarity oils are esters. Esters are thecovalent compounds formed between acids and alcohols. In otherembodiments, the medium polarity oils are fatty acid esters which arecompounds formed between fatty acids and alcohols. In still otherembodiments, the medium polarity oils are glyceryl esters. Glycerylesters are primarily fatty acid mono-, di-, and/or tri-glycerides. Onesuch glyceryl ester is glyceryl caprylate/caprate. Glycerylcaprylate/caprate is available from the Abitec Company under the tradename CAPMUL® MCM, NF and from Sasol Olefins & Surfactants GmbH under thetrade name IMWITOR 742. Glyceryl caprylate/caprate is also known by itssynonyms: caprylic/capric glycerides (INCI name); mono- anddi-glycerides (NF name); glycerol monocaprylocaprate; medium chain mono-& diglycerides; glycerides C8-10 mono- di- tri-; and glyceryl mono- &dicaprylo/caprate. Glyceryl caprylate/caprate has an octanol-waterpartition coefficient value of 1.21 as determined experimentally by theASTM method mentioned above. Glyceryl caprylate/caprate is amono-diglyceride of medium chain fatty acids (mainly caprylic andcapric). It is a mixture of monoacylglycerols, mainlymono-O-octanoylglycerol and mono-O-decanolyglycerol, containing variablequantities of di- and triacylglycerols. It is obtained by directesterification of glycerol with caprylic (octanoic) and capric(decanoic) acids. CAPMUL MCM, NF and IMWITOR 742 meet the requirementsof the USP/NF under the NF monograph for “mono- and di-glycerides.”

TABLE 1 Medium Polarity Oil Log P value Resorcinol 0.80* Anisyl Alcohol1.10* Benzoic Acid 1.87* Benzyl Alcohol 1.10* Ethyl Acetate 0.73* EthylGallate 1.30* Phenyl ethanol 1.42* Phenoxyethanol 1.16* Propyl Gallate1.80* Glyceryl Caprate/Caprylate  1.21** *value obtained from ExploringQSAR, Vol. 2 **value determined experimentally by ASTM method

The concentrations of the medium polarity oil and the antibacterialagent components in the compositions are at amounts that exhibitsynergistic antibacterial activity against bacterial biofilms. Theconcentration of the medium polarity oil in the compositions can varywith different oils, but generally can be 1 to 50% w/w, or 1 to 40% w/w,or 1 to 30% w/w, or 1 to 25% w/w, or 1 to 20% w/w, or 1 to 10% w/w, or 5to 10% w/w, or 5 to 11% w/w, or 5 to 12% w/w, or 5 to 15% w/w, or 5 to25% w/w, or 5 to 30% w/w, or 5 to 40%, w/w, or 5 to 50% w/w, or 6 to 50%w/w, or 6 to 40% w/w, or 6 to 30% w/w, or 6 to 25% w/w, or 6 to 20% w/w,or 6 to 15% w/w, or 6 to 12% w/w, or 6 to 11% w/w, or 6 to 10% w/w, or 7to 50% w/w, or 7 to 40% w/w, or 7 to 30% w/w, or 7 to 25% w/w, or 7 to20% w/w, or 7 to 15% w/w, or 7 to 12% w/w, or 7 to 11% w/w, or 7 to 10%w/w, or 8 to 50% w/w, or 8 to 40% w/w, or 8 to 30% w/w, or 8 to 25% w/w,or 8 to 20% w/w, or 8 to 15% w/w, or 8 to 12% w/w, or 8 to 11% w/w, or 8to 10% w/w, or 9 to 50% w/w, or 9 to 40% w/w, or 9 to 30% w/w, or 9 to25% w/w, or 9 to 20% w/w, or 9 to 15% w/w, or 9 to 12% w/w, or 9 to 11%w/w, or 9 to 10% w/w, or at least 5% w/w, or at least 6% w/w, or atleast 7% w/w, or at least 8% w/w, or at least 9% w/w, or at least 10%w/w. In some embodiments, the medium polarity oil is an ester, fattyacid ester, or glyceryl ester and the concentration in the compositionis 6 to 50% w/w, or 6 to 40% w/w, or 6 to 30% w/w, or 6 to 25% w/w, or 6to 20% w/w, or 6 to 15% w/w, or 6 to 12% w/w, or 6 to 11% w/w, or 6 to10% w/w, or 7 to 50% w/w, or 7 to 40% w/w, or 7 to 30% w/w, or 7 to 25%w/w, or 7 to 20% w/w, or 7 to 15% w/w, or 7 to 12% w/w, or 7 to 11% w/w,or 7 to 10% w/w, or 8 to 50% w/w, or 8 to 40% w/w, or 8 to 30% w/w, or 8to 25% w/w, or 8 to 20% w/w, or 8 to 15% w/w, or 8 to 12% w/w, or 8 to11% w/w, or 8 to 10% w/w, or 9 to 50% w/w, or 9 to 40% w/w, or 9 to 30%w/w, or 9 to 25% w/w, or 9 to 20% w/w, or 9 to 15% w/w, or 9 to 12% w/w,or 9 to 11% w/w, or 9 to 10% w/w, or at least 6% w/w, or at least 7%w/w, or at least 8% w/w, or at least 9% w/w, or at least 10% w/w. Insome embodiments, the medium polarity oil is glyceryl caprate/caprylateand the concentration in the composition is 6 to 50% w/w, or 6 to 40%w/w, or 6 to 30% w/w, or 6 to 25% w/w, or 6 to 20% w/w, or 6 to 15% w/w,or 6 to 12% w/w, or 6 to 11% w/w, or 6 to 10% w/w, or 7 to 50% w/w, or 7to 40% w/w, or 7 to 30% w/w, or 7 to 25% w/w, or 7 to 20% w/w, or 7 to15% w/w, or 7 to 12% w/w, or 7 to 11% w/w, or 7 to 10% w/w, or 8 to 50%w/w, or 8 to 40% w/w, or 8 to 30% w/w, or 8 to 25% w/w, or 8 to 20% w/w,or 8 to 15% w/w, or 8 to 12% w/w, or 8 to 11% w/w, or 8 to 10% w/w, or 9to 50% w/w, or 9 to 40% w/w, or 9 to 30% w/w, or 9 to 25% w/w, or 9 to20% w/w, or 9 to 15% w/w, or 9 to 12% w/w, or 9 to 11% w/w, or 9 to 10%w/w, or at least 6% w/w, or at least 7% w/w, or at least 8% w/w, or atleast 9% w/w, or at least 10% w/w.

B. Antibacterial Agents

The compositions of the invention comprise at least one antibacterialagent. Various antibacterial agents are suitable for use with thepresent invention. Suitable antibacterial agents include silvercompounds such as the following non-limiting examples: elemental silver,silver nanoparticles, silver zeolite, silver sulfadiazine, ionizedsilver, and silver salts such as silver chloride and silver nitrate.Other suitable antibacterial agents include iodine compounds such as thefollowing non-limiting examples: iodine, tincture of iodine, Lugol'siodine solution, iodides, iodine topical solution, iodine complexed withphosphate ester of alkylaryloxy polyethylene, iodoquinol, undecoyliumchloride-iodine, nonylphenoxypolyethanol-iodine complex, and iodophorssuch as povidone-iodine (PVP-iodine), polyvinyl alcohol-iodine,polyvinyl oxazolidone-iodine, polyvinyl imidazole-iodine, polyvinylmorpholone-iodine, and polyvinyl caprolactam-iodine,nonylphenolethoxylate-iodine, soluble starch-iodine,betacyclodextrin-iodine, polyoxyethylenepolyoxypropylenecondensate-iodine, ethoxylated linear alcohol-iodine, andcadexomer-iodine. Additional non-limiting examples of suitableantibacterial agents include: quaternary ammonium compounds such asbenzalkonium chloride, benzethonium chloride, methylbenzethoniumchloride, cetalkonium chloride, cetylpyridinium chloride, cetrimonium,cetrimide, dofanium chloride, tetraethylammonium bromide,didecyldimethylammonium chloride and domiphen bromide; chlorinecontaining compounds such as sodium hypochlorite, calcium hypochlorite,and chlorine dioxide; hydrogen peroxide; benzoic acid and its salts;benzoyl peroxide; benzyl alcohol; bispyrithione salts; boric acid;camphorated metacresol; camphorated phenol; chlorobutanol; cloflucarban;dapsone; dehydroacetic acid and its salts; ethyl alcohol;hexachlorophene; hexitidine; hexylresorcinol; hydroxybenzoic acid andits salts; isopropyl alcohol; mafenide acetate; magnesium pyrithione;merbromin; mercufenol chloride; methylparaben; metronidazole and itsderivatives; mupirocin and its salts; nitrofurazone; n-Propanol; organicperoxides; p-chloro-m-xylenol; phenol; phenoxyethanol; phenyl alcohol;phenyl ethyl alcohol; selenium sulfide; sodium oxychlorosene; sodiumsulfacetamide; sorbic acid and its salts; sulfur;tetrachlorosalicylanilide; thymol; tribromsalan; triclocarbon;triclosan; and zinc pyrithione.

Antibiotics and antibacterial peptides are also suitable antibacterialagents. Suitable antibiotics include polypeptide antibiotics, examplesof which are colistin (polymyxin E), colistin A (polymyxin E1), colistinB (polymyxin E2), colistin sulfate, colistimethate sodium, actinomycin,bacitracin, and polymyxin B. Other suitable antibiotics includeaminoglycoside antibiotics, examples of which are gentamicin, gentamicinsulfate, neomycin, kanamycin, and tobramycin. Other suitable antibioticsinclude glycopeptide antibiotics, examples of which are vancomycin,teicoplanin, telavancin, ramoplanin, decaplanin, and bleomycin. Othersuitable antibiotics include macrolide antibiotics, examples of whichare azithromycin, clarithromycin, erythromycin, fidaxomicin,telithromycin, spiramycin, and troleandomycin. Other suitableantibiotics include mupirocin, calcium mupirocin, and retapamulin.

In some embodiments, the antibacterial agent is an iodine compound. Inother embodiments, the iodine compound is an iodophor. In still otherembodiments, the iodophor is cadexomer-iodine or povidone-iodine. Insome embodiments, the cadexomer-iodine is at a concentration in thecomposition of 40 to 60% w/w, or 40 to 50% w/w, or 45 to 55% w/w, or 50to 60% w/w, or about 50% w/w. In some embodiments, the povidone-iodineis at a concentration in the compositions of 1 to 25% w/w, or 1 to 20%w/w, or 1 to 15% w/w, or 3 to 15% w/w, or 5 to 10% w/w, or about 5% w/w,or about 10% w/w. In some embodiments, the antibacterial agent is asilver compound. In other embodiments, the silver compound is silversulfadiazine, silver nitrate, or silver chloride. In some embodiments,the silver sulfadiazine is at a concentration in the composition of 0.1to 10% w/w, or 0.1 to 5% w/w, or 0.1 to 2% w/w, or 0.1 to 1.5%, or 0.5to 5% w/w, or 0.5 to 2% w/w, or 0.5 to 1.5% w/w, or 0.5 to 1% w/w, or0.1 to 1% w/w, or 1 to 5% w/w, or about 0.5% w/w, or about 1% w/w. Insome embodiments, the silver nitrate is at a concentration in thecomposition of 0.1 to 10% w/w, or 0.1 to 5% w/w, or 0.1 to 2% w/w, or0.1 to 1.5%, or 0.5 to 1% w/w, or 0.5 to 5% w/w, or 0.5 to 2% w/w, or0.5 to 1.5% w/w, or 0.1 to 1% w/w, or 1 to 5% w/w, or about 0.5% w/w, orabout 1% w/w. In some embodiments, the silver chloride is at aconcentration in the composition of 0.1 to 10% w/w, or 0.1 to 5% w/w, or0.1 to 2% w/w, or 0.1 to 1.5%, or 0.5 to 1% w/w, or 0.5 to 5% w/w, or0.5 to 2% w/w, or 0.5 to 1.5% w/w, or 0.1 to 1% w/w, or 1 to 5% w/w, orabout 0.5% w/w, or about 1% w/w. In some embodiments, the antibacterialagent is an antibiotic. In other embodiments, the antibiotic is anaminoglycoside antibiotic. In still other embodiments, theaminoglycoside antibiotic is gentamicin or gentamicin sulfate. In someembodiments, the gentamicin or gentamicin sulfate is at a concentrationin the composition of 0.1 to 10% w/w, or 0.1 to 5% w/w, or 0.1 to 2%w/w, or 0.1 to 1% w/w, or 0.5 to 5% w/w, or 0.5 to 2% w/w, or 0.5 to 1%w/w, or 0.5 to 0.7% w/w, or 0.7 to 1% w/w, or about 0.7% w/w. In otherembodiments, the antibiotic is a polypeptide antibiotic. In still otherembodiments, the polypeptide antibiotic is colistin or colistin sulfate.In some embodiments, the colistin or colistin sulfate is at aconcentration in the composition of 0.01 to 5% w/w, or 0.01 to 2% w/w,or 0.01 to 1% w/w, or 0.01 to 0.5% w/w, or 0.01 to 0.2% w/w, or 0.05 to1% w/w, or 0.05 to 0.5% w/w, or 0.05 to 0.2% w/w, or 0.05 to 0.15% w/wor about 0.1% w/w. In some embodiments, the antibacterial agent is notchlorhexidine gluconate.

The concentrations of the medium polarity oil and the antibacterialagent components in the compositions are at amounts that exhibitsynergistic antibacterial activity against bacterial biofilms. Theconcentration of the antibacterial agent in the compositions can varywith different antibacterial agents, but generally can be 0.01 to 75%w/w, or 0.01 to 60% w/w, or 0.01 to 50% w/w, or any range or numbertherein (e.g., at least 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, and up to 75 wt. %).

C. Manufacture

The compositions of the invention may be manufactured by methods andequipment known in the art for manufacture of topical products andproducts designed for application to non-biological surfaces, such asmedical devices. Such methods include, but are not limited to the use ofmechanical mixers including LIGHTNIN propeller mixers; COWLESdissolvers; SILVERSON dispersers; counter-rotating side-scrappingmixers; homogenizers and dispersers, including in-line or in-tankrotor-stator homogenizers; and mills, including 3-roll mills, ointmentmills, or rotor-stator mills. “All-in-one” vacuum mixing systems thathave a rotating side-scrapping mixer plus an in-tank homogenizer mayalso be used. Such mixers include, but are not limited to OLSA mixers,FRYMA-KORUMA mixers, and LEE TRI-MIX TURBO-SHEAR kettles. Thecompositions of the invention can be manufactured from small laboratoryscale batches to full-scale production batches.

II. BACTERIAL BIOFILMS

The compositions of the invention are suitable for the reduction ofbacteria in and/or elimination of both gram-positive and gram-negativebacterial biofilms. Non-limiting examples of gram-positive bacteriainclude Staphylococcus spp., such as Staphylococcus aureus, methicillinresistant Staphylococcus aureus (MRSA), and Staphylococcus epidermidis;Streptococcus spp, such as Streptococcus pneumonia; Bacillus spp.;Listeria monocytogenes; enterococci spp.; and lactic acid bacteria, suchas Lactobacillus plantarum and Lactococcus lactis. Non-limiting examplesof gram-negative bacteria include Pseudomonas spp., such as Pseudomonasaeruginosa; and Escherichia coli.

A. In-Vitro Biofilm Model

An in-vitro biofilm model was used to evaluate the biofilm efficacy ofthe formulations of the invention against bacterial biofilms. Bacteriaare spotted onto a collagen matrix resting on a filter on a blood agarplate and incubated to allow biofilm formation. The model mimics in-vivowound biofilms in that nutrients are provided from below the biofilmwhile topical treatments are applied at the air interface above. Thisin-vitro model and methodology is disclosed in the poster presentation,A Versatile In Vitro Biofilm Model Using Two Wound Pathogens to ScreenFormulations, Van der Kar, et al., presented at the 2010 Wound HealingSociety Annual Meeting, Poster BRC09, on Apr. 18, 2010 in Orlando, Fla.,and is herein incorporated by reference. Further in-vitro biofilm modelsand methodologies are disclosed in the following publications all ofwhich are herein incorporated by reference: Penetration of Rifampinthrough Staphylococcus epidermidis Biofilms, Zheng, et al.,Antimicrobial Agents and Chemotherapy, March 2002, p. 900-903; OxygenLimitation Contributes to Antibiotic Tolerance of Pseudomonas aeruginosain Biofilms, Borriello et al., Antimicrobial Agents and Chemotherapy,July 2004, p. 2659-2664; and Heterogeneity in Pseudomonas aeruginosaBiofilms Includes Expression of Ribosome Hibernation Factors in theAntibiotic-Tolerant Subpopulation and Hypoxia-Induced Stress Response inthe Metabolically Active Population, Williamson et al., Journal ofBacteriology, February 2012, p. 2062-2073.

III. METHODS OF USE AND TREATMENT

The compositions of the invention are useful for the reduction ofbacteria in and/or elimination of bacterial biofilms on biological andnon-biological surfaces, and are also useful for treatment of wounds,skin lesions, mucous membrane lesions, and other biological surfacesinfected or contaminated with bacterial biofilms.

A. Biological Surfaces

The compositions of the invention are useful for reducing bacteria inand/or eliminating a bacterial biofilm on a biological surface byadministering the compositions to the biological surface. Non-limitingexamples of biological surfaces include wounds (including chronic andacute wounds), skin lesions, skin, mucous membranes, mucous membranelesions, internal organs, body cavity, oral cavity, bone tissue, muscletissue, nerve tissue, ocular tissue, urinary tract tissue, lung andtrachea tissue, sinus tissue, ear tissue, dental tissue, gum tissue,nasal tissue, vascular tissue, cardiac tissue, epithelium, andepithelial lesions, and peritoneal tissue. Non-limiting examples ofchronic wounds include diabetic foot ulcers, venous ulcers, arterialulcers, decubitus ulcers, stasis ulcers, pressure ulcers, and burns.Non-limiting examples of acute wounds include cuts and surgical wounds.Non-limiting examples of skin lesions and mucous membrane lesionsinclude blisters, ulcers, abrasions, warts, scrapes, and skin andmucosal infections such as staph or MRSA infections. Examples of skinlesions and mucous membrane lesions are disclosed in “Description ofSkin Lesions”, MacNeal, Robert J., the on-line Merck Manual ProfessionalVersion, March 2013,http://www.merckmanuals.com/professional/dermatologic-disorders/approach-to-the-dermatologic-patient/description-of-skin-lesionsherein incorporated by reference. Skin lesions can appear on theepidermis, lips, ear canal, scalp, cuticle, nail bed, or genitalia.Mucous membrane lesions can appear on the oral mucosa, nasal mucosa,penile and vaginal mucosa, or anus.

B. Topical Treatment of Wounds

The compositions of the invention are useful for the treatment ofwounds, including chronic wounds and acute wounds, infected orcontaminated with bacterial biofilms, by topically administering thecompositions to the wound. Non-limiting examples of chronic woundsinclude diabetic foot ulcers, venous ulcers, arterial ulcers, decubitusulcers, stasis ulcers, pressure ulcers, and burns. Non-limiting examplesof acute wounds include cuts and surgical wounds.

C. Topical Treatment of Skin Lesions and Mucous Membrane Lesions

The compositions of the invention are useful for the treatment of skinlesions or mucous membrane lesions infected or contaminated withbacterial biofilms by topically administering the compositions to theskin lesion or mucous membrane lesions. Non-limiting examples of skinlesions and mucous membrane lesions include blisters, ulcerations,abrasions, warts, scrapes, and skin and mucosal infections such as staphor MRSA infections. Skin lesions can appear on the epidermis, lips, earcanal, scalp, cuticle, nail bed, or genitalia. Mucous membrane lesionscan appear on the oral mucosa, nasal mucosa, penile and vaginal mucosa,or anus.

D. Treatment of Other Biological Surfaces

The compositions of the invention are useful for the treatment of otherbiological surfaces infected or contaminated with bacterial biofilms byadministering the compositions to the biological surface. Non-limitingexamples of other biological surfaces include internal organs, bodycavity, oral cavity, bone tissue, muscle tissue, nerve tissue, oculartissue, urinary tract tissue, lung and trachea tissue, sinus tissue, eartissue, dental tissue, gum tissue, nasal tissue, vascular tissue,cardiac tissue, epithelium, and epithelial lesions, and peritonealtissue.

E. Non-Biological Surfaces

The compositions of the invention are useful for reducing bacteria inand/or eliminating a bacterial biofilm on a non-biological surface, suchas a medical device, by administering the compositions to thenon-biological surface. Non-limiting examples of medical devices includeurinary tract prostheses; urinary tract catheters, peritoneal membranecatheters, peritoneal dialysis catheters, indwelling catheters forhemodialysis and for chronic administration of chemotherapeutic agents(Hickman catheters); cardiac implants such as pacemakers, prostheticheart valves, ventricular assist devices, and synthetic vascular graftsand stents; prostheses; percutaneous sutures; and tracheal andventilator tubing. The surface of an article of manufacturing, includingmedical devices, can be coated with the compositions of the inventionsprior to the presence of a bacterial biofilm in order to prevent theformation of bacterial biofilms; or can be coated after the presence ofa bacterial biofilm on the surface in order to reduce bacteria in and/oreliminate the bacterial biofilm on the surface.

IV. EXAMPLES A. Example 1 Determination of Octanol-Water PartitionCoefficient for CAPMUL MCM by ASTM Method

1. Prepared reference standard samples of compounds with known log Pvalues shown in Table 2 at concentrations of approximately 200 mg/L inmethanol.

2. Prepared test sample of CAPMUL MCM at a concentration ofapproximately 200 mg/mL in methanol.

3. Ran reference and test samples on HPLC using the parameters shown inTable 3.

4. Compared the retention times of the reference standards to theretention time of CAPMUL MCM to calculate the log P value of CAPMUL MCMas per ASTM method.

The retention times of the reference substances are shown in Table 2 andplotted in FIG. 2. The retention time of CAPMUL MCM is 3.011 giving alog P of 1.21 for CAPMUL MCM.

TABLE 2 log Reference retention Substance log P time Anisyl alcohol 1.10.5008 Phenoxyethanol 1.16 0.5161 Diethyl phthalate 2.42 0.5944 Benzylcinnamate 4.06 0.8331 Benzyl salicylate 4.31 0.8719 Dibutyl sebacate 6.31.2709

TABLE 3 Mobile Phase Solvent A 40:60 ACN/H2O Mobile Phase Solvent B70:30 ACN/H2O Detector 249 nm Injection 20 microliters Flow Rate 1mL/min Column Luna C18(2) 5 micron, 100 angstroms, 250 × 4.6 mmGradient: 0-15 min 100% A 15-15:50 Increase to 100% B 15:50-20 100% B20-25 100% A Temperature 22° C.

B. Example 2 Formulations

Various formulations were prepared and are shown in tables 4-9 below.

Cadexomer iodine based formulations are shown in Table 4.

TABLE 4 Cadexomer Iodine Based Formulations Formula Cadexomer CadexomerCadexomer Cadexomer/ Cadexomer Cadexomer Iodine Iodine/ Iodine/Cadexomer 10% oil Iodine/ Iodine Component (% w/w) Control 5% oil 10%oil Control control 2.5% oil 1% oil PEG-400 38 34.3 30.4 41 30.4 36 37.2PEG-4000 10 9 8 10.7 8 9.5 9.8 Poloxamer 184 2.1 1.8 1.6 2.2 1.6 1.9 1.9CAPMUL MCM NF — 5 10 — 10 2.5 1 Cadexomer Iodine 50 50 50 — — 50 50Cadexomer Base — — — 46 50 — —

Procedure (for concentration of each ingredient, see Table 4.): Mixedall ingredients except cadexomer iodine and/or cadexomer base at 70° C.until uniform. Added cadexomer iodine or cadexomer base and mixed untiluniform. Cooled to room temperature (RT) while mixing.

Silver chloride based formulations are shown in Table 5.

TABLE 5 Silver Chloride Based Formulations Component Formula (% w/w) Agalone Oil alone Ag + Oil HEC 250 HX (Aqualon) 11 11 11 Silver Chloride 1— 1 CAPMUL MCM — 10 10 Phenoxyethanol 0.7 0.7 0.7 PEG 600 39 35 34 PEG400 39 35 34 PEG 3350 4 4 4 ARISTOFLEX AVC 1 1 1 Glycerin 4 4 4

Procedure (for concentration of each ingredient, see Table 5.):Homogenized PEG 600, PEG 400, PEG 3350, Glycerin, ARISTOFLEX AVC, CAPMULMCM (if present), and Silver Chloride (if present) at high temperatureusing a Silverson homogenizer for 1 minute at 8000 rpm. Cooled themixture to 50° C. and added HEC 250 HX while mixing. Continued mixinguntil the temperature was less than 35° C.

Other silver based formulations are shown in Table 6.

TABLE 6 Other Silver Based Formulations Formula 1% AgNO3/ 1% AgCl/ 1%SSD/ 1% 1% 1% Component (% w/w) 9% oil 9% oil 9% oil AgNO3 AgCl SSDPlacebo Poloxamer 407 6 6 6 6 6 6 7 Glycerin 2 2 2 2 2 2 2 StearylAlcohol 3.8 3.8 3.8 3.8 3.7 3.7 3.0 Polysorbate 60 3.7 3.7 3.7 3.8 3.73.7 3.2 CAPMUL MCM NF 9 9 9 — — — 9 Isopropyl Myristate 4.6 4.6 4.6 13.713.7 13.7 4.6 Silver Nitrate 1 — — 1 — — — Silver Chloride — 1 — — 1 — —Silver Sulfadiazine — — 1 — — 1 — PHOSPHOLIPON 90G 2.3 2.3 2.3 2.3 2.32.3 2.3 DI Water qs ad 100 qs ad 100 qs ad 100 qs ad 100 qs ad 100 qs ad100 qs ad 100

Procedure (for concentration of each ingredient, see Table 6.):Poloxamer 407, glycerin and water were mixed until dissolved at RT toform a water phase. Stearyl alcohol, polysorbate 60, isopropylmyristate, PHOSPHOLIPON G and CAPMUL MCM (if present) were mixed at 70°C. until clear to form an oil phase. The water phase and oil phase werecombined and mixed at 70° C. for 2 hours and then cooled to RT whilemixing. An active phase was made with water (10% w/w) and silver nitrateor silver chloride or silver sulfadiazine (SSD). The active phase(except for placebo) was then was added to the batch and mixed untiluniform.

Povidone-iodine based formulations are shown in Table 7.

TABLE 7 Povidone-Iodine Based Formulations Formula 5% Component Placebo5% PVI + (% w/w) plus oil PVI 10% oil Poloxamer-407 15.0 15.1 14.6Propylene Glycol 5.0 5.0 5.4 Povidone-Iodine — 5.0 4.9 CAPMUL MCM NF10.0 — 10.1 DI Water qs ad 100 qs ad 100 qs ad 100

Procedure (for concentration of each ingredient, see Table 7.):Poloxamer-407 and propylene glycol were dissolved in water. PovidoneIodine and/or CAPMUL MCM were added while mixing and mixed untilhomogenous. The formulations containing povidone-iodine were brownsolutions

Gentamicin based formulations are shown in Table 8.

TABLE 8 Gentamicin Based Formulations Formula 0.7% Component Placebo0.7% GENTA + (% w/w) plus oil GENTA 10% oil Poloxamer-407 15.0 14.8 15.0Propylene Glycol 5.0 5.0 5.0 Gentamicin Sulfate — 0.7 0.7 CAPMUL MCM NF10.0 — 10.8 DI Water qs ad 100 qs ad 100 qs ad 100

Procedure (for concentration of each ingredient, see Table 8.):Poloxamer-407 and propylene glycol were dissolved in water. Gentamicinsulfate and/or CAPMUL MCM were added while mixing and mixed untilhomogenous. The formulations containing CAPMUL MCM were thick, ringinggels. The viscosity of the ringing gel formulation “0.7% GENTA+10% oil”was 89,000 cps as measured using a Brookfield RV viscometer with a smallsample adapter, spindle #14, at room temperature (22°-25 ° C.), at 10rpm for 1 minute.

Colistin based formulations are shown in Table 9.

TABLE 9 Colistin Based Formulations Formula 0.1% 0.1% Colistin ComponentPlacebo Colistin Sulfate + (% w/w) plus oil Sulfate 10% oilPoloxamer-407 15.0 15.0 15.0 Propylene Glycol 5.0 5.0 5.1 ColistinSulfate — 0.1 0.1 CAPMUL MCM NF 10.0 — 10.0 DI Water qs ad 100 qs ad 100qs ad 100

Procedure (for concentration of each ingredient, see Table 9.):Poloxamer-407 and propylene glycol were dissolved in water. Colistinsulfate and/or CAPMUL MCM were added while mixing and mixed untilhomogenous. The formulations containing CAPMUL MCM were thick, ringinggels.

C. Example 3 In-Vitro P. aeruginosa Biofilm Model with Various TreatmentFormulations

P. aeruginosa ATCC 27312 was grown overnight on tryptic soy agar (TSA)at 37° C. The next day, a single colony was picked and passed intotryptic soy broth (TSB), then grown at 37° C. overnight with shaking(150 rpm). The overnight culture was diluted to ˜1.5×10⁸ cfu/mL in PBS(inoculum). Tryptic soy agar with 5% sheep blood (TSAB) plates wereprepared with six 13 mm black 0.2 micron TEFLON filters. Each 13 mmfilter had a single 4 mm collagen plug placed in the center and was theninoculated with 3 μL of inoculum placed on the center of the plug (13 mmfilter plus inoculated plug=colony biofilm assembly or CBFA). The CBFAplates were transferred to a 37° C. incubator and incubated for 24hours. At the end of the incubation, growth was sampled and treatmentwith the test formulations of Example 2 was started. The testformulations for the cadexomer iodine based formulations (Table 4) weremixed 50/50 (weight/volume) with PBS and applied (200 μL) to PBSmoistened (200 μL) 13 mm TELFA non-adherent dressing squares. The othertest formulations (gel and liquid formulations from Tables 5-9) wereapplied directly to the PBS moistened TEFLA squares (the liquidformulations were mixed for 10 seconds prior to the application). Thetreatments were applied with the formulation directly in contact withthe biofilm (TELFA on top) and gently tamped down to ensure contact withthe biofilm (moist control was TELFA only). The treated CBFA plates weretransferred to the 37° C. incubator and incubated for 24 hours. At theend of the incubation, the treated CBFA were recovered into 5 mL DEBroth PBS and vortexed at 2500 rpm for 2 minutes to knock off thetreatments and resuspend any surviving bacteria. The recoveries wereserially diluted (1:10 eight point dilution series in PBS broth) and 10volumes spotted on Charcoal Agar plates (the plates serve to neutralizeany active treatments). The plates were allowed to dry and incubated at37° C. overnight with colony counts determined the next day. Counts wereconverted to colony forming units per milliliter and transformed to logvalues. Efficacy was determined by subtracting the log cfu/mL value of atreatment from the moist control to generate a log reduction value inaddition to direct comparison of recovered log cfu/mL.

The results of the log reduction of bacteria in the biofilm model aftertreatment with the treatment formulations from Example 2 (cadexomeriodine based formulations—Table 4) vs. moist control are shown in FIG.3. The results of the log reduction of bacteria in the biofilm modelafter treatment with the treatment formulations from Example 2 (silverchloride based formulations—Table 5) vs. moist control are shown in FIG.4. Results of the log reduction of bacteria in the biofilm model aftertreatment with the treatment formulations from Example 2 (other silverbased formulations—Table 6) vs. moist control are shown in FIG. 5. Theresults of the log reduction of bacteria in the biofilm model aftertreatment with the treatment formulations from Example 2(povidone-iodine based formulations—Table 7) vs. moist control are shownin FIG. 6. The results of the log reduction of bacteria in the biofilmmodel after treatment with the treatment formulations from Example 2(gentamicin based formulations—Table 8) vs. moist control are shown inFIG. 7. The results of the log reduction of bacteria in the biofilmmodel after treatment with the treatment formulations from Example 2(colistin based formulations—Table 9) vs. moist control are shown inFIG. 8.

The results in FIG. 5 show the cumulative log reduction effect of the“1% AgCl” formulation plus the “Placebo” formulation was less than thelog reduction effect of the “1%AgCl/9% oil” formulation; the cumulativelog reduction effect of the “1% AgNO” formulation plus the “Placebo”formulation was less than the log reduction effect of the “1% AgNO/9%oil” formulation; and the cumulative log reduction effect of the “1%SSD” formulation plus the “Placebo” formulation was less than the logreduction effect of the “1% SSD/9% oil” formulation. These resultsindicate synergistic antibacterial activity by the combination of theantibacterial agent and the medium polarity oil.

The results in FIG. 4 show the cumulative log reduction effect of the“Ag alone” formulation plus the “Oil alone” formulation is less than thelog reduction effect of the “Ag+Oil” formulation at 24 hours indicatingsynergistic antibacterial activity by the combination of theantibacterial agent and the medium polarity oil.

The results in FIG. 3 show the cumulative log reduction effect of the“Cadexomer 10% oil control” formulation plus the “Cadexomer IodineControl” formulation is less than the log reduction effect of the“Cadexomer Iodine 10% oil” formulation indicating synergisticantibacterial activity by the combination of the antibacterial agent andthe medium polarity oil.

The results in FIG. 6 show the cumulative log reduction effect of the“Placebo plus oil” formulation plus the “5% PVI” formulation is lessthan the log reduction effect of the “5% PVI+10% oil” formulationindicating synergistic antibacterial activity by the combination of theantibacterial agent and the medium polarity oil.

The results in FIG. 7 show the cumulative log reduction effect of the“Placebo plus oil” formulation plus the “0.7% GENTA” formulation is lessthan the log reduction effect of the “0.7% GENTA+10% oil” formulationindicating synergistic antibacterial activity by the combination of theantibacterial agent and the medium polarity oil.

The results in FIG. 8 show the cumulative log reduction effect of the“Placebo plus oil” formulation plus the “0.1% colistin sulfate”formulation is less than the log reduction effect of the “0.1% colistinsulfate+10% oil” formulation indicating synergistic antibacterialactivity by the combination of the antibacterial agent and the mediumpolarity oil.

1-114. (canceled)
 115. A method of treating a wound, mucous membranelesion, or skin lesion infected or contaminated with a bacterialbiofilm, the method comprising topically administering to the wound,mucous membrane lesion, or skin lesion a composition consistingessentially of a pharmaceutical carrier and a sole anti-bacterial agent,wherein the anti-bacterial agent consists of a combination of a mediumpolarity oil having an octanol-water partition coefficient (log P) of0.5 to 2.0 and at least one iodine compound, wherein the medium polarityoil is benzyl alcohol or propyl gallate and is at a concentration of4.5% w/w to 5.5% w/w, and wherein the combination consisting of themedium polarity oil and the iodine compound produces a synergisticantibacterial effect against bacteria in the biofilm.
 116. The method ofclaim 115, wherein the iodine compound is an iodophor.
 117. The methodof claim 116, wherein the iodophor is selected from povidone-iodine(PVP-iodine), polyvinyl alcohol-iodine, polyvinyl oxazolidone-iodine,polyvinyl imidazole-iodine, polyvinyl morpholone-iodine, polyvinylcaprolactam-iodine, nonylphenolethoxylate-iodine, soluble starch-iodine,betacyclodextrin-iodine, polyoxyethylenepolyoxypropylenecondensate-iodine, ethoxylated linear alcohol-iodine, orcadexomer-iodine.
 118. The method of claim 116, wherein the iodophor iscadexomer-iodine.
 119. The method of claim 118, wherein theconcentration of the cadexomer-iodine is 40% w/w to 60% w/w.
 120. Themethod of claim 115, wherein the pharmaceutical carrier is a lotion,solution, suspension, liquid, emulsion, cream, gel, ringing gel,ointment, paste, aerosol spray, aerosol foam, non-aerosol spray,non-aerosol foam, film, or sheet.
 121. The method of claim 115, whereinthe bacterial biofilm is a gram-positive bacterial biofilm.
 122. Themethod of claim 121, wherein the gram-positive bacterium isStaphylococcus aureus or methicillin resistant Staphylococcus aureus(MRSA).
 123. The method of claim 115, wherein the bacterial biofilm is agram-negative bacterial biofilm.
 124. The method of claim 123, whereinthe gram-negative bacterial biofilm is Pseudomonas aeruginosa.
 125. Themethod of claim 115, wherein the wound is a chronic wound.
 126. Themethod of claim 125, wherein the chronic wound is a diabetic foot ulcer,venous ulcer, arterial ulcer, decubitus ulcer, stasis ulcer, pressureulcer, or burn.
 127. The method of claim 115, wherein the mucousmembrane lesion or skin lesion is a blister, ulceration, abrasion, wart,scrape, or infection.